| It has been more than thirty years since the identification of Human Immunodeficiency Virus-1 (HIV-1) and remarkable advances were obtained in understanding the pathogenesis and immunology of HIV-1. However, an effective vaccine against HIV-1 has not yet been developed and major scientific obstacles still remain. HIV-1 has an extraordinary diversity and has evolved multiple mechanisms to evade immune responses. Improving the cellular immune response and inducing broadly neutralizing antibody (NAb) are two important directions in the development of HIV-1 vaccine. The potency and persistance of cellular immune responses were mainly affected by immunogen and immunomodulatory factor in vivo. As an important immune-regulating factor, IL-21 plays a pleiotropic effect on regulating immune response, since IL-21 receptor is broadly expressed on various immune cells. Hence, IL-21 has been widely used in the study of vaccine as an important adjuvant to improve immune response. Nowadays, strain-specific neutralizing antibodies could be induced by HIV-1 vaccines in some studies, but broadly NAbs and persistent cellular immune responses were much more difficult to elicit by the current immune strategy. However, cross-reactive neutralizing antibodies have been found in 10% ～25% HIV-1 infected individuals, indicating that the immune system could induce broadly NAbs in the long-term struggle with HIV-1. Therefore, identifying broadly NAbs from HIV-1 infected individuals and exploring their mature processes are effective strategy to study the mechanism of protective humoral immune response. The results will guide rational vaccine design to induce broadly NAb. Meanwhile, as a kind of immunotherapeutic agents, broadly NAb could be used to directly confer protection by passive immunity. Focusing on the above two important directions in the development of HIV-1 vaccine, we have accomplished two parts in the present study.1. Evaluation of IL-21 adjuvant on regulating cellular immune responses in HIV-1 vaccinated mice.By analyzing the immune responses induced by HIV-1 DNA vaccine prime-vaccina virus/protein vaccine boost in mice, we found that this immune strategy could induce broad and lasting antigen-specific antibody responses (IgG, IgM, IgA, IgG1, IgG2a, IgG2b and IgG3) and cellular immune responses, including specific cytokine secretion (IFN-γ, IL-2 and TNF-α) and multifunctional T cells in HIV-1 vaccinated mice. Based on this immune strategy, mouse IL-21 adjuvant was co-immunized with HIV-1 vaccines to evaluate the effects of mIL-21 on regulating cellular immune responses. In this study, we found that mIL-21 could adjust the efficacy of HIV-1 vaccine through increasing the Thl type immune response. The ability to secrete HIV-1 specific IFN-y and TNF-a was improved in CD4+ T and CD4+ TEM cells. Mouse IL-21 could increase the frequencies of specific multifunctional CD4+ T and CD4+ TEM cells. Meanwhile, the frequency of NK cells was increased by administration of mIL-21.2. Identification of monoclonal broadly NAbs from Chinese HIV-1 infected donors and explore the mechanisms of their mature processes.On the base of collected samples from Chinese HIV-1 infected donors who displayed cross-reactive neutralizing activity, we identified a series of monoclonal neutralizing antibodies with limited breadth. We analyzed NAb mature process in a donor by studying samples from 2006 to 2009. In cooperation with the Scripps Research Institute (TSRI) scientists, we isolated a neutralizing antibody named DRVIA7 using antigen-specific single B cell sorting and monoclonal antibody expression technology. DRVIA7 was a CD4 binding site (CD4bs) directed antibody with modest neutralizing breadth (33% on Global Panel,36% on DRVI Panel). Gene sequence analysis revealed that the heavy chain of DRVIA7 was IgHV 1-02*02 allelic origin with a SHM (somatic hypermutation) of 19%, and light chain was IgKV-1-05*03 allelic origin with a SHM of 17%, indicating that DRVIA7 may belong to the VRC01-like antibodies.The crystal structure of DRVIA7 was determined by TSRI scientists, which confirmed that DRVIA7 was a VRC01-like antibody. The N terminus and LCDR1 of DRVIA7 light chain were found to clash with V5 and N276 glycans on gp120 by the crystal structural in comparison with VRC01-gp120 complex. Deglycosylation could increase the binding capacity of DRVIA7 with gp120, suggesting the clash between glycans on gp120 and antibody light chain reduced the binding capacity and may restrict the development of neutralizing activity.Based on the above structural analysis, the N-terminal truncation of DRVIA7 light chain and utilization VRC01 LCDR1 could significantly improve the neutralizing breadth (100% on Global Panel,72% on DRVI Panel), verifying the clash between glycans on gp120 and antibody light chain restricted the development of neutralizing activity. Redesigned antibody could neutralize VRC01-resistant virus BJOX2000. When DRVIA7 heavy chain was paired with VRC01 light chain, the antibody acquired a broad neutralizing activity similar to VRC01 (100% vs.92% on Global Panel,92% vs.96% on DRVI Panel) and could neutralize four VRC01-resistant virus (BJOX2000, DU172,57128,3817). However, the development of DRVIA7 light chain was not completed.In cooperation with TSRI scientists, we studied the antibody repertoires via deep sequencing and bioinformatics analysis. DRVIA7-lineage genes were selected from longitudinal antibody repertoires in three time points to verify the neutralizing activity in vitro. We found that DRVIA7-lineage genes should emerge shortly before 2006, peaked in 2008, and began to decline in 2009. Though DRVIA7-lineage heavy chains had not been found in 2006, selected heavy chains which should be functional precursors showed broadly neutralizing activity (Global Panel:1.25 μg/ml,93%; DRVI Panel:0.84 μg/ml,86%). Selected heavy chains from 2008 (Global Panel:0.92 μg/ml,90%; DRVI Panel:0.74 μg/ml,92%) and 2009 (Global Panel:0.54 μg/ml, 98%; DRVI Panel:0.36 μg/ml,96%) displayed increasing neutralizing breadth and potency when paired with VRC01 light chain. DRVIA7-lineage heavy chains could rapidly mature within 2 years. Though light chains from 2006 had already displayed VRCO1-like LCDR3 signature, only one light chain displayed broadly neutralizing activity (Global Panel:6.62 μg/ml,75%; DRVI Panel:2.27 μg/ml,60%) in 2009 when paired with DRVIA7 heavy chain. The results indicated that the immune system tried to overcome this barrier. By pairing heavy with light chains from DRVI01, we acquired a series of more broadly functional antibodies (83% vs.33% on Global Panel). Though paired heavy and light chains in vitro may not originate from the same B cell in vivo, more potent and broadly neutralizing antibody may emerge in future driven by the evolution of immune system.In conclusion, we described the effect of mIL-21 on improving cellular immune response, which provided rationale for further preclinical studies involving IL-21 as a potential immune-based cytokine adjuvant. In addition, for the first time, we isolated and identified a monoclonal broadly neutralizing antibody from a Chinese HIV-1 infected donor. We demonstrated that key glycans on gp120 formed barriers to the elicitation of bNAbs and revealed how this lineage antibody emerged and matured in vivo, which may guide the rational design of HIV-1 vaccine. Key glycans on gp120 should be considered in vaccine designing to this targeting site, and broadly NAbs might be induced by activating antibody precursors. We also acquired a series of monoclonal broadly NAbs, which may be used as immunotherapeutic agents in future. This research provided impetus on the development of HIV-1 vaccine in two important goals (to improve cellular immune response and induce broadly neutralizing antibody) to some extent. |
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